Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability

The outer membrane of gram-negative bacteria functions as a permeability ba rrier that protects cells against a large number of antibacterial agents. O prL protein of Pseudomonas putida has been shown to he crucial to maintain the stability of this cell component (J. J. Rodriguez-Herva, M.-I. Ramos-Go nzalez, and J. L. Ramos. J. Bacteriol. 178:1699-1706, 1996). In the present study we cloned and mutagenized the orf1, role, tolR, toll, and tolB genes from P. putida KT2440, which were located upstream of the oprL gene. Polar and nonpolar mutations of the P. putida tale, tolR, tolA, and tolB genes w ere generated in vitro by using the Omega-Km(r) interposon, which carries t wo transcriptional stop signals, or a promoterless xylE cassette, lacking a ny transcriptional stop signal, respectively. The mutant constructs were us ed to inactivate, by reverse genetics procedures, the corresponding chromos omal copies of the genes. The phenotype of each mutant strain was analyzed and compared with those of the wild-type strain and the previously characte rized P. putida oprL::xylE mutant. All mutant strains exhibited a similar p henotype: altered cell morphology, bleb formation at the cell surface, rele ase of periplasmic and outer membrane proteins to the extracellular medium, increased sensitivity to a variety of compounds (i.e., EDTA, sodium dodecy l sulfate, deoxycholate, and some antibiotics), filament formation, and sev erely reduced cell motility. Altogether, these results demonstrate the impo rtance of the Tol-OprL system for the maintenance of outer membrane integri ty in P. putida and suggest a possible role of these proteins in assembling outer membrane components.